Molecular diagnostics in virology

Journal of Clinical Virology 31 (2004) 239–247

Review

Molecular diagnostics in virology

Guy Vernet∗

Advanced Technology Unit, bioMerieux, 69280 Marcy-l’Etoile, France

Accepted 11 June 2004

Abstract

Molecular biology has significantly improved diagnosis in the field of clinical virology. Virus discovery and rapid implementation ofdiagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. Viral load and antiviralresistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus(HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and CMV. It will be important to add to this panel assays for other viruses ofthe herpesviridaefamily. Qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organtransplantation. Screening of other blood-borne viruses (parvovirus B19, HAV), multiplexing of detection and test automation to improve

nosisade torcialeal-timequired

40

0401

practicability and reduce costs will be the next steps. A major evolution in the near future will be the generalization of NAT for the diagof viral etiology in patients, mostly with respiratory, CNS or gastro-intestinal diseases. Major technical improvements have been mavoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. Commeoffers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. Ramplification has allowed the development of new NAT platforms but automation and integration of all steps of the reaction are still reto reduce hands-on-time, time-to-result and costs, and to increase throughput.© 2004 Elsevier B.V. All rights reserved.

Keywords:Virology; Diagnostics; Nucleic acid tests

Contents1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. New, emerging or re-emerging viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.1. Viral safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2. Viral disease management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3. Technical constraints and recent improvements of molecular assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
242242

243245245

246

2

2.4. Automation of molecular assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5. Regulatory considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.6. Reagents for the diagnosis of viral infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.7. DNA-microarrays in clinical virology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8. Treatment monitoring. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

∗ Tel.: +33 4 78 87 24 08; fax: +33 4 78 87 53 40.E-mail address:[email protected] (G. Vernet).

1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jcv.2004.06.003

240 G. Vernet / Journal of Clinical Virology 31 (2004) 239–247

1. Introduction

Molecular biology has revolutionized all domains ofviruses diagnosis including the rapid identification of emerg-ing or re-emerging viruses, viral safety of blood productsor organ transplants and viral disease management. One ofthe major driving forces for the introduction of moleculartechniques in virology has been the absence of easy andperforming multiplication techniques similar to those devel-oped for bacteriology. The most striking illustration of thepower of molecular techniques concerns blood transmittedviruses—human immunodeficiency virus (HIV), hepatitis Bvirus (HBV) and hepatitis C virus (HCV) for which spectacu-lar progresses in the detection and treatment of viral diseaseshave been made following the introduction of qualitative andquantitative nucleic acid tests (NAT). The recent discovery ofa new human coronavirus responsible for severe acute respi-ratory syndrome (SARS) epidemic is another example. It isobvious that NAT will encourage the development of antivi-ral drugs which, compared to antibiotics, has been delayedpartly because performing efficiency assessment techniqueswere lacking.

2. New, emerging or re-emerging viruses

gensh withv ed ac no d toi fer-e anh andT m-p anso tionp gen-e neum sso-c one

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have allowed the characterization of very important humanpathogens like HHV-8, responsible of Kaposi sarcoma orHCV which induce acute or chronic hepatitis, cirrhosis andhepatocarcinoma. However, other viruses detected in a simi-lar way are still waiting for the demonstration of their clinicalimportance. This illustrates the need to verify Koch’s postu-late and the importance of keeping laboratory competenciesfor classical virology—tissue culture, electron microscopyand animal experiments—which plays a major role in virusdiscovery, together with epidemiological studies. Large epi-demiological studies are also required to assess the clinicalinterest of new pathogens.

Molecular diagnostics have been very rapidly imple-mented in clinical virology laboratories following the dis-covery of hMPV (van den Hoogen et al., 2001; Peiris et al.,2003; Boivin et al., 2003) and SARS-CoV (Ksiazek et al.,2003; Drosten et al., 2003; Anderson, 2003). A prospectivestudy on the prevalence of hMPV could be initiated as earlyas during the 2000–2001 winter, although the virus has beendiscovered in 2000 only. There has been only a few weekslag between sequencing SARS-CoV and availability of thefirst commercial NAT.

2.1. Viral safety

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During the last 15 years, many new human pathoave been discovered among which eight were virusesarious pathogenicity. Molecular techniques have playentral role in their discovery (Table 1). The constructiof cDNA libraries by cloning techniques has been use

dentify HCV and hepatitis E Virus. Representational difnce analysis (RDA) was successful in identifying humerpes virus 8 (HHV-8), and the hepatic viruses GBV-CTV. RDA allows the detection of viral sequences by coaring whole nucleic acids sequences in cells from humr animals before and after infection. Reverse-trancripolymerase chain reaction (RT-PCR) with random or derate primers has been used to identify human metapovirus (hMPV), the virus SEN and the coronavirus a

iated to SARS (SARS-CoV). Molecular techniques al

able 1ole of molecular biology in virus discovery

ear Virus Method of discovery

988 Hepatitis E Animal transmission + cDNlibrary

989 Hepatitis C Random primed cDNA libra995 Human herpes virus 8 RDA995 GBV-C RDA997 TT virus RDA001 Human Metapneumovirus Tissue culture + RT-PCR

random primers001 SEN virus RT-PCR with degenerate

primers

DA: representational difference analysis.

-

NAT are more and more used to exclude blood dnations from patients infected by viruses (Allain, 2003).HCV and HIV testing has been implemented as partroutine screening in blood banks in 14 and 7 Europcountries respectively. In France, two commercial offerProcleix (Gen-Probe, Inc. USA) and NucliSens Extrac(bioMerieux, France) associated with Cobas Ampliscre(Roche Diagnostics, Switzerland)—are used to scrdonations for HIV and HCV infections. This allowed threduction of residual risk from 1 in 400,000 to 1 in 2millions for HIV and 1 in 760,000 to 1 in 5 millions for HCV(Assal et al., 2003). However, there is room from improvement as few contaminated blood units are still missedto the lack of sensitivity induced by pooling strategies. Cconstraints must also be considered if further improvemeare to be considered. Cost effectiveness of HIV and HNAT addition to serology testing is already very low:USA it has been calculated that the cost of each saved li4.7–11.2 millions US$ per year (Jackson et al., 2003).

Hepatitis B virus screening using molecular bioloshould also be included as even the most recent antigesays (HbsAg) assays miss infected blood units. As anample, single-sample HBV testing would allow the detetion of 35–50 additional contaminated units among 107 unitstested (Biswas et al., 2003). Monovalent or trivalent assay(HIV, HBV, HCV) are proposed by Roche Diagnostics (Ampli NAT) and Gen-Probe (Procleix Ultrio) but blood unishould not be pooled to provide sufficient sensitivity. Pcleix Ultrio detects single seroconversions 20 days earthan Abbott Prism-HBsAg but only 13 days in pools of 8 a11.5 days in pools of 24 (Cambie, 2002).

G. Vernet / Journal of Clinical Virology 31 (2004) 239–247 241

Alternatively, an ultracentrifugation step could be intro-duced following pooling to obtain a higher sensitivity com-pared to current antigen assays (Roth et al., 2002).

Screening for West-Nile virus contamination has beenimplemented in US blood banks. From late June to mid-September 2003, approximately 2.5 million donations werescreened. Twelve hundred eighty-five (0.05%) were initiallyreactive for WNV by using nucleic acid-amplification testsand 601 (0.02% of the total donations) are considered pre-sumptive viremic donations (i.e. a donation that is repeatedlyreactive by the primary and/or alternate NAT assay or a pri-mary NAT assay with a very high signal) (CDC, 2003).Otherviruses (parvovirus B19, Hepatitis A virus) are also trans-mitted by blood donation and may be part of the screeningin a near future. However, NAT may not always be the bestdiagnosis approach and antigen tests or antibody tests maybe efficient and less expensive alternatives.

Automation and integration of NAT is necessary to reducecosts and quarantine delays for blood units supply. In thisrespect, the recent approval by US Food and Drug Admin-istration (FDA) of the TIGRIS molecular diagnostic system(Gen-Probe, San Diego, USA) is a major breakthrough as ithas been designed to process 500 samples in 8 h.

Integration and time-to-result are also very important pa-rameters to be considered to insure viral safety of transplanto tantt n byH

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Gardner et al. (2003)have deduced from sequence alignmentsthat a real-time TaqMan assay should contain not less thannine primer and probe sets to detect with the same sensitivityall HIV strains in a geographically representative panel. To re-duce the impact of variability on amplification and detectionefficiency, one can use primers and probes with 2′ O-methylbases, degenerate bases or “universal” bases, such as inosineor nebularine. Touchdown PCR protocols, in which the an-nealing temperature slightly decreases during the successiveamplification cycles to bracket the melting temperature Tmof the reaction, provides sensitivity even when primers havemismatches with target sequences of divergent species in aviral family. Finally, degenerate primers or probes, with mix-tures of the bases found in sequence databases among variousspecies, may be useful to detect all species of a viral family.

It is often desirable to provide the capability for paneldetection, i.e. to detect several viruses that can be responsi-ble for a disease. For example, Coyle et al. have describedat the winter meeting of European Society for Clinical Vi-rology (Copenhagen, January 2004) a molecular viral res-piratory strip for the detection of 12 common respiratoryviruses. Whenever possible, consensus primers able to detectall viruses from a family or genus must be used. There areseveral examples of such consensus primers for enterovirus(Kammerer et al., 1994), flavivirus (Scaramozzino et al.,2 ra ustb ible,t lti-p ndi-v bet s ind s. Fore ssay( T-P B,R r of< n thev ntifyc SVB ta).T d ast n ofa es,n l con-d Real-t ringt tor ve-l de-t rs ofp rigi-n s fori ackso e ofl ual

rgans, especially lung, heart and liver. It is very imporo determine the status of transplants regarding infectioIV, HBV, HCV and viruses from theherpesviridaefamily.

.2. Viral disease management

NAT have significantly improved identification of viruss etiologic agents of human diseases affecting variouans, especially respiratory and gastro-enteric tracts and

ral nervous system (CNS). As a consequence, rapid anreatments can be initiated and considerably reduce moty and mortality as, for example, in the case of herpesephalitis. The increasing number of available antiviral dill even accentuate the need for positive viral identificatimilarly, unnecessary antibiotic treatments can be avor reduced and hospitalisation durations shorten. Treatmf chronic viral diseases are very efficiently monitorediral load assays.

However there are still obstacles that prevent a wideremination of molecular assays.

.3. Technical constraints and recent improvements ofolecular assays

The extreme genetic variability of some viruses, eially RNA viruses (which RNA-polymerases have no proeading activity), often makes their diagnosis difficult. Tost striking examples are found in the norovirus famhich contains viruses responsible for the vast majoritastro-intestinal epidemics in adults. HIV diagnosis isuite difficult to achieve due to its high genetic variabil

001) orHerpesviridae(Tenorio et al., 1993). However, theibility to amplify all viruses with the same efficiency me carefully evaluated. If such an approach is not poss

wo different possibilities exist for panel detection: mulex detection in single tubes or parallel detection in iidual tubes. Mixing primers in a single amplification tuo achieve multiplex detection of viruses usually resultecreased sensitivity of assays compared to single testxample, we have observed, using a DNA-microarray asee below), that the analytical sensitivity of multiplex RCR detection of six viruses, i.e. influenza A, influenzaSV A/B, parainfluenza 1, 2 and 3 is reduced by a facto1–2 logs compared to single detections, depending oirus. Nevertheless, this multiplex assay was able to ideorrectly 21/22 infections in respiratory specimen (one Rinfection was misidentified as RSV A; unpublished da

he formation of primer dimers is generally considerehe major cause of sensitivity loss but careful optimisatioll parameters of amplification including primer, enzymucleotides and salts concentrations as well as protocoitions are required to obtain expected performances.

ime assays (see below) that monitor signal apparition duhe amplification step are also limited in their capacityealize multiplex detection by the number of available waengths in existing equipments which currently allows theection of three viruses only. Instead of mixing several pairimers in a single tube, nucleic acids purified from the oal clinical specimen can be distributed into several tube

ndependent amplification and detection. Major drawbf this approach are the reduction of sensitivity becaus

ower amounts of nucleic acid available for each individ


amplification, higher hands-on times required to manipulateall the different tubes, difficulty to automate the distributionof small volumes of purified nucleic acids without introduc-ing cross-contaminations and higher costs due to the need forenzymes in each tube.

Internal controls (IC) are important components to mon-itor each step of the assay from extraction of nucleic acidsto detection. Because inhibitors of the amplification reaction,which are frequent in some specimen types, will also impactits amplification, the presence of an IC is a strong valida-tion in case of negative result.Niesters (2002)has describedan original approach for internal control of NAT: the use ofviral universal controls that can be added to each specimenand be amplified with specific primers, preferably in a multi-plex format with primers for the virus to detect. Seal herpesvirus 1 and phocine distemper virus can be used to controlNAT for DNA and RNA viruses respectively. Of course, ani-mal viruses which can not infect humans are required. Otherstrategies for IC involve synthetic materials, i.e. plasmidsor transcripts which contain sequences able to bind the testprimers and a specific probe.

2.4. Automation of molecular assays

Clinical virology laboratories have high expectations int .

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have also been designed for real-time assays like TaqMan(Fig. 1A) or molecular beacons (Fig. 1B) in which a quenchermolecule is removed from the vicinity of the fluorescentmarker upon binding to RNA or DNA generated duringamplification cycles. In the FRET technology (Fig. 1C),two probes, one with a fluorescence donor and one witha fluorescence acceptor molecule are designed to bindadjacent sequences of the amplified material to generatesignal (Mackay et al., 2002). Real-time techniques have beendesigned for PCR or NASBA amplification and have severaladvantages which facilitate automation and reduce time-to-result (30–120 min) and hands-on-times. They are performedin closed devices which do not need to be opened to transferamplified material for end-point detection, thus reducingthe risk of laboratory or samples cross-contamination. Theuse of real-time platforms makes the general organisation ofmolecular biology laboratories easier by reducing the con-straints on activities segregation in different rooms to controlcontaminations.Table 3shows existing real-time automates.

The next generation of NAT platforms will integrate sam-ple preparation, amplification and detection in a single testdevice GeneXpert (CEPHEID, USA) is the first fully inte-grated system which allows the detection ofBacillus an-thracis or group B streptococcus in approximately 45 mindirectly from clinical specimen. Several viral assays basedo blef

hed

2

de-v urcesn ssaysa oft reg-u itrod aret rdingt lini-c ations proval

erm of automation and integration of molecular assaysA major bottleneck in the workflow of these laborato

s at the level of sample preparation.Table 2shows autoated systems for nucleic acid purification that are curre

ommercialised. Most of them use the nucleic acid binroperties of silica (Boom et al., 1990). As many as 96 samles can be handled in a single run on the BioRobot 9Qiagen GmbH, Germany). Time-to-result and, more imant, hands-on-time tend to decrease, with the most rquipments requiring no longer than 20 min of techni

ime for more than 24 samples.New labelling technologies that do not need solid-ph

eparation have allowed the development of real-olecular assays in which the detection of ampliconone as soon as they appear during amplification. Theimple real-time detection chemistry uses the SYBR gye which specifically binds during double-stranded Denerated during PCR. Several probe technologies (Fig. 1)

able 2utomated systems for the purification of nucleic acids

anufacturer Technology Instr

iagen Magnetic particles/silica extraction BioMagnetic particles/silica extraction BioR

oche diagnostics Magnetic particle/specific oligonucleotidecapture probes

MagNA

Filter-based/silica extraction Amp

ioMerieux Magnetic particles/silica extraction NucMagnetic particles/silica extraction Nuc

(in dev

Samples/run Run duration Hands on tim

9604 96 <2.5 h 20 minZ1 1–6 20 min

32 (capillaries or plates) 2 h/24 samples 35 min/24 sam

72 2.5 h 30 min/24 sam

extractor 10 45 min 20 minEasyMagnt)

24 1 h <20 min

n real-time NASBA on this platform will soon be availarom bioMerieux.

Several IVD companies offer commercial NAT for tiagnosis of viral diseases.

.5. Regulatory considerations

Besides technical difficulties, another obstacle to theelopment of molecular assays is the importance of resoeeded to optimise, produce and validate home-brew and to build up documentation required for qualification

echniques and laboratories. New European Communitylations will even increase this need. It is the role of in viagnostic (IVD) companies to provide reagents which

he results of careful optimisation and are produced accoo high quality manufacturing procedures. They have cal and regulatory affairs departments that conduct validtudies and assemble documentation required to get ap


Fig. 1. Probes used for real-time detection. FromMackay et al., 2002. A: TaqMan 5′-nuclease oligoprobes. As the DNA-polymerase progresses along theamplicon, it displaces and hydrolyses the oligoprobe via its 5′–3′ endonuclease activity. When the reporter group (R) is removed from the amplicon it is nolonger under the inhibition effect of the quencher group (Q). B: molecular beacons. Hybridisation of the complementary part of the probe on the ampliconseparates the fluorophore (F) from the quencher group (Q). C: LightCycler FRET probes. Two probes, one with a donor group (D) and one with an acceptorgroup (A) bind in adjacent positions on the amplicon. The donor group transfers the energy acquired upon excitation to the acceptor groups which generatesfluorescence.

of the reagents. However, even for IVD companies, the con-ception and validation process is time-consuming and time-to-market may be long. This is especially a problem whena diagnostic tool is urgently needed in case of emergenceof a new virus. One possibility to reduce time-to-market isto release “research use only” (RUO) assays or assays thathave the “CE analytical” approval in Europe or the status of“analyte specific reagent” (ASR) in the USA. In this case,commercial products that have excellent analytical sensitiv-ity and are manufactured according to quality standards ofthe IVD industry can be used by clinical virology labora-tories, which have the responsibility to validate their use asdiagnostic tools and obtain authorization to use them.

Table 3Real-time amplification platforms

Manufacturer Platform Number of samples Number of wavelengths

Applied ABI PRISM 7700 96 Continuous wavelength detection from 500 to660 nm allows the use of multiple fluorophoresin a single reactionBiosciences ABI PRISM 7900 394

Roche diagnostics LightCycler 32 3COBAS TaqMan 48 24 per processing block 4

2 processing blocksbioMerieux EasyQ 48 4Stratagene Mx3000/Mx4000 96 4BC chann

locks cC

on).

2.6. Reagents for the diagnosis of viral infections

Infections by HIV and hepatitis B and C viruses are usu-ally well diagnosed using serology. Only diagnosis of earlyprimo-infections may benefit from NAT. Platforms like Am-plicor Amplicor from Roche Diagnostics or EasyQ frombioMerieux that are usually used for viral load measurementduring therapy (see below) are also suitable for the early de-tection of HIV or HCV infections. Many other infectionsand especially acute infections for which IgM appear onlyseveral days after onset of symptoms cannot be efficientlydiagnosed using serology assays. Forty to sixty percent ofcommunity acquired pneumonia that require hospitalisation

io-Rad iCycler iQ 96epheid Smart Cycler II 16 independent

1–6 processing bepheid/bioMerieuxa GeneXpert 4a Integrated real-time platform (sample preparation and amplificati

4els per processing block,an be interconnected

4

4


Table 4QCMD programs in 2002 (fromWallace, 2003)

Program type Number ofparticipants

Number ofdata sets

Total tests Correct (%) Equivocal (%) False +ve (%) False−ve (%) Commercialassays (%)

CMV 89 105 1260 81.6 1.0 2.9 20.3 81.0EBV 61 67 603 80.6 0.0 3.0 19.0 29.9Enterovirus 93 100 1200 79.0 1.6 5.5 27.7 93.0HBV 70 97 776 89.2 0.1 2.1 11.9 51.5HCV 80 124 992 91.8 0.1 0.0 9.2 88.0HIV 64 90 720 84.8 0.6 0.0 14.6 90.0HSV 98 109 1308 87.8 0.9 0.9 14.8 9.2

have no known aetiology despite intensive investigation andthis percentage is even higher when less severe lower respi-ratory tract infections (LRTI) are considered (L. Kaiser, per-sonal communication). In a recent study,Henrickson et al.(2004), have shown, using multiplex RT-PCR that 40% ofchildren hospitalised for LRTI are infected with the sevenmost common respiratory viruses. Similarly, a recent surveyof encephalitis leading to hospitalisation in the USA from1988 to 1997 has revealed that nearly 60% had no aetiol-ogy (Khetsuriani et al., 2002). Absence of specific antiviraltreatments for most viruses, which limits prescription of bio-logical tests and weak performances of diagnostic tests basedon viral culture or serology are major explanations of this sit-uation.

NAT, which have been implemented by many large Euro-pean hospitals, significantly improve viral diagnosis. How-ever, there are several obstacles to the generalization ofmolecular diagnostics in smaller, decentralized laboratories.

A major obstacle is the fact that, except for HIV, HBV,HCV and CMV, virology NAT are most often home-brewassays which sometimes suffer from bad performances andpoor batch to batch consistency. However, quality of NAT is

Table 5Commercially available virology NAT (other than HIV, HBV, HCV)

csASBA

R

H mRNA

N

A

PO

I

improving as illustrated by results of the 2002–2003 Qual-ity Control Molecular Diagnostics (QCMD) proficiency test-ing (Table 4). Panels for different viruses have been sent to61–111 European laboratories. The percentage of correct re-sults ranged from 79% for enterovirus to 91.8% for HCV.The percentage of false-positive results which reflects labo-ratory or cross-contaminations and used to be high, droppedto 5.5% (Wallace et al., 2003).

Table 5shows some products currently commercialisedby major companies for viruses other than HIV, HBV andHCV, although the list may not be exhaustive. Most of themare ASR or RUO kits although some are EC marked. Themajority of these reagents have been designed to run on real-time platforms.

Results shown by Liolios et al. in 2001 illustrate the in-terest of multiplex detection of respiratory viruses by NAT.One hundred forty-three clinical specimen were tested usingthe Hexaplex assay from Prodesse Inc. (USA) which detectssix viruses in a single tube using PCR and detection withmicroplate capture and peroxidase-labelled probes. 9 sam-ples were detected with the Prodesse assay only and not withimmunofluorescence or viral culture (Table 6).

Argene biosoft Artus GmbH bioMerieuxPCR Real-time PCR Real-time N

espiratory viruses NA Coronavirus,Sars-CoV

Sars-CoV

erpesviridae HSV 1 and 2(generic andtyping)

HSV 1 and 2,EBV, VZV

CMVq (pp67

eurotropic viruses Enterovirus Enterovirus,West-Nilevirus

Enterovirus

denovirus NA NA NA

apilloma virus NA NA NAthers NA Dengue virus,

parvovirusB19

NA

VD: in vitro diagnostic. The letter “q” means a quantitative assay.

Digene Corp. Prodesse Inc. Roche diagnostiPCR PCR and real-time

PCRReal-time PCR

NA Hexaplex (InfluenzaA and B,parinfluenza A, 2and 3, RSV A and B,hMPV

Sars-CoV

) NA Herpes multiplex(HSV 1 and 2, CMV,EBV, VZV, HHV6)

HSV 1 and 2, EBVq,

NA NA NA

NA Adenoplex:(adenovirus A-F)

NA

HPV (IVD assay) NA HPV (IVD assay)NA NA Parvovirus B19, HAVq


Table 6Multiplex Nat for the detection of respiratory viruses (Hexaplex, Prodesse Inc.)

RT-PCR/viral culture and/or immunofluorescence PIV-1 PIV-2 PIV-3 Inf A Inf B RSV

+/+ 0 0 0 6 1 1+/− 0 0 1 7 1 00−/− 143 143 142 130 141 142−/+ 0 0 0 0 0 0

FromLiolios et al., 2001.

2.7. DNA-microarrays in clinical virology

DNA-microarrays or DNA-Chips are very powerful detec-tion tools that can be combined with amplification techniquesto detect viruses or virus variants (reviewed inClewley,2004). Wang et al. (2002)have described a microarray spot-ted with 70-mer oligonucleotides which represent the fivemost conserved sequences (more than 20/70 bases are con-served among all representative sequences in a virus sequencealignment) of each virus of interest. The Chip contains 1600different probes. Following PCR amplification of genetic ma-terial in the clinical specimen using random primers, hybridi-sation onto the microarray was able to identify respiratoryviruses in the enterovirus, rhinovirus, paramyxovirus, aden-ovirus and herpesvirus families. However, this technique hasnot yet been extensively validated for routine diagnosis in aclinical virology laboratory.

We have developed assays combining RT-PCR andDNA-microarrays for the detection of viruses. The arraysare manufactured using the photolithography in situ syn-thesis technology (Affymetrix, USA) and contain 20-meroligonucleotides. Sequence signatures are identified usingextensive sequence databases because they are conserved inall viruses of a genus or family or in all subtypes or isolatesof a virus and are not found in other viruses. For each baseo andp arep neart ensup irus,h nda tionP toryo edue lep asers ribeda ajorv a Aa nicalsd es.

rus( etec-t dic-t r of

HPV types that are more or less closely associated to cervi-cal cancer is high. DNA-microarrays may thus be appropriatefor the multiplex detection of all these types. Such an assayhas been described byAn et al. (2003)and is distributed bybiomedlab Co. (South-Korea). It is based on a consensus am-plification and on 30-mer probes that are able to detect anddiscriminate 22 HPV types and has shown an association of92.5–97.5% between HPV positivity and lesions of differentseverity or carcinoma whereas HPV infection was only foundin 35.1% of cases when cytology was normal.

2.8. Treatment monitoring

Viral load platforms are available from several IVD com-panies (Versant from Bayer Diagnostics; Cobas Ampliprep/Amplicor from Roche Diagnostics, MiniMag/NucliSensEasyQ from bioMerieux). Significant progress have beenmade in the ability of most HIV assays to detect all sub-types of HIV-1 but no commercial assay exist for HIV-2.The analytical sensitivity of HBV viral load assays should beincreased to reach the same performances as those of HIVassays, especially in the case of infections by variants withlow replication competencies. For efficient treatment moni-toring of immunosuppressed patients, for example in the caseof organ transplantation, viral load assays should also be de-v andH

arep ghha CVs g tech-n Vi-rk ucha How-e ocel-l bed oesn n of ah veh or-p tionsm

typ-ih n with

f the signature, probes perfectly matching the targetrobes with a mismatch at the interrogated positionresent on the array. If polymorphisms are present in or

he signature, variant probes may also be present. Consrimers have been designed for enterovirus, flaviverpesviridae, parainfluenza and influenza virus, RSV adenovirus. They can be combined in multiplex detecCR or RT-PCR assays for the diagnosis of viral respirar CNS infections. Following amplification, DNA is labellsing a newly developed diazomethyl chemistry (Laayount al., 2003). A complete line of instruments (sampreparation, thermocycler, hybridisation station and lcanner) is available to perform the assay. As descbove, a respiratory assay designed to detect six miruses (parainfluenza 1, 2 and 3, RSV A/B, influenznd B) in a single specimen has demonstrated high cliensitivity in preliminary evaluation.Fig. 2 illustrates theiscriminatory capacity of this technology for CNS virus

Assays for the identification of human papilloma viHPV) are commercialised by several companies. The dion of a highly pathogenic HPV type has a very high preive value for cervical carcinoma. However, the numbe

s

eloped for Epstein-Barr virus, Varicella-Zooster VirusHV6.Genotyping tests are now commercially available and

art of the biological follow-up of treated patients althouome-brew assays are still used in most laboratories (Korn etl., 2003). HIV and HBV resistance assays as well as Hubtyping assays are generally based on the sequencinology (Trugene HIV and HBV from Bayer HealthCare;oSeq HIV-1 from Celera Diagnostics; 5′ genotyping HCVit from Bayer healthcare). Hybridisation techniques, ss LiPA assays (Innogenetics, Belgium) are also used.ver, the number of probes that can be spotted on nitr

ulose strips is limited and only few polymorphisms canetected which is convenient for HCV subtyping but dot for resistance tests which are based on the detectioigh number of mutations. In addition, HIV and HBV haighly variable genomes and naturally occurring polymhisms that are present in the vicinity of resistance mutaay affect the binding efficiency of probes.DNA-microarrays are an alternative for resistance or

ng reagents for viruses or bacteria (Vernet et al., 2004). Weave developed an assay based on RT-PCR and detectio


Fig. 2. bioMerieux DNA-microarray for the detection of neurotropic viruses. Following nucleic acid purification, amplification is performed by PCR usinga single touchdown protocol in three tubes, one forherpesviridae(one primer pair for HHV 1, 2, 3, 5 and 6), one for enteroviruses (one primer pair forall serotypes) and one for flaviviruses (one primer pair for all viruses). Amplification products are combined and labelled using diazomethyl chemistry andhybridised on a DNA-microaaray which contains 20-mer oligonucleotides. Two or four probes are used for the detection of each base of sequence signaturesdetermined for each virus. A total of 20,000 probes are synthesised on this DNA-microarray which has been designed for the simultaneous detection of virusesfrom theherpesviridaefamily and from the flavivirus, enterovirus, paramyxovirus, poliomavirus, bunyavirus and orthopoxvirus genus. A: amplicons generatedresolved using the bioanalyzer (Agilent Technologies). B: image of the DNA-microarray obtained with a confocal laser reader. C: resolution capability of thearray. Closely related viruses hybridise with very different efficiency on heterologous probes.

high density probe arrays, designed to detect 204 antiretrovi-ral resistance mutations simultaneously in gag cleavage sites,protease, reverse transcriptase, integrase and gp41. This as-say has been tested on a panel of 99 HIV-1 patients on atotal of 4465 relevant codons in comparison with the clas-sic sequence-based method. Key resistance mutations werecorrectly identified in 95 and 92% of codons in protease andreverse transcriptase, respectively (Gonzalez et al., 2004).We have also developed a similar assay for the detection ofpolymorphisms in the complete HBV genome: 78 antiviralresistance mutations in pol gene, 146 vaccine, diagnostic orimmunotherapy mutation in s gene and 209 mutations in basiccore promoter, pre-core, core, X, pre-S1 and pre-S2 regionsthat may have an impact on disease evolution or treatment ef-ficiency. This assay is currently under evaluation in the frameof HepBVar a European collaborative group for the study ofemerging variants of Hepatitis B virus.

3. Conclusion

In the coming years, more and more laboratories will offerto clinicians viral diagnosis based on nucleic acid tests. Manybiological and instrumentation problems that have slowed thegeneralization of molecular assays have been resolved buts r im-

provements are expected in the integration and automation ofNAT diagnostic platforms to reduce hands-on-time, time-to-result and costs and to increase throughput. IVD companieshave engaged in development programs to provide clinicalvirologists with equipments and application menus adaptedto their diagnosis needs.

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